Vol. 168, No. 3, 1990 May 16, 1990

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 1297-1302

CULTURED VASCULAR SMOOTH MUSCLE CELLS PROM PORCINE CORONARY ARTERY POSSESSAl AND A2 ADENOSINE RECEPTOR ACTIVITY

Ira ~illsl andHenry Gewirtz

Division of Cardiology, RhodeIsland Hospital, andBrown University Programin Medicine, Providence, RhodeIsland 02903

Received

March

26,

1990

SUMMARY: This study tested the hypothesisthat an Al adenosinereceptor capable of inhibiting adenylatecyclase activity is presentin porcine coronary vascular smoothmuscle cells. In the absenceof blockade of the A2 adenosinereceptor, the Al adenosinereceptor agonistsphenylisopropyladenosine(PIA) and cyclopentyladenosine(CPA) (lo-9M) failed to inhibit Gpp(NH)p stimulatedadenylatecyclase activity. However, after blockade of the A2 adenosinereceptor with 30 nM CGS 15943A, cyclopentyladenosine(lo-9M) inhibited Gpp(NH)p stimulated adenylate cyclase activity by 27+3% (4.3kO.7, Mean+SEM; pmoles/min/mgvs 5.9+0.8, Pc.05). The data demonstratethat both Al and A2 adenosine receptors are present in coronary vascular smooth muscle. The results indicate that adenosinemay mediate both vasodilation and vasoconstriction in the coronary circulation via A2 and Al adenosinereceptors,respectively. 01990 Academic Press, Inc. Previous studies utilizing cultured aortic vascular smooth muscle cells have demonstratedadenosinestimulation of adenylate cyclase activity via the A2 adenosine receptor (1). Although there are datafrom a study employing an intact caninemodel which suggestthat an Al adenosinereceptor alsomay be presenton coronary smoothmuscle(2), the questionremainsunsettled. The presenceof Al adenosinereceptor mediatedinhibition of adenylate cyclase has not been demonstratedin a pure culture of coronary vascular smooth muscle. The purpose of the present study, therefore, was to test the hypothesis that an Al adenosinereceptor, capableof mediatinginhibition of adenylatecyclase activity, is presentin porcine coronary vascular smoothmusclecells. MATERIALS AND METHODS Smooth Muscle Culture: Cultured vascular smooth muscle cells were prepared as describedby Gunther et al. (3). Coronary arterieswere obtained from pigs freshly killed

1 To whom correspondenceshouldbe addressed. ABBREVIATIONS USED: PIA, phenylisopropyladenosine;CPA, cyclopentyladenosine; Gpp(NH)p, 5’-guanylyl-imidodiphosphate. 0006-291X/90 1297

$1.50

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either at a local slaughterhouseor in the laboratory. Adventitia wasdissectedaway and the endothelium scrapedoff with a scalpelblade. The vesselswere cut into small piecesand digestedin Ml99 containing 2 mg/ml collagenase,0.250 mg/ml elastaseand 0.5% fetal calf serum. Tissue digestion was carried out in a gently shakingwater bath for 90 minutesat 37’C. At the conclusionof the digestionperiod, 7 mls of Ml99 containing 10% fetal calf serumwas added. The digestion mixture was titurated ten times in a 20 ml syringe with a three inch 12 gauge needle. The mixture was then filtered through a 100 urn Cellector Tissue Sieve (Vineland, NJ) after which the filtrate was centrifuged at 200 x g for 5 minutes. Harvested cells were resuspendedin 10 mls of Ml99 (containing 10% fetal calf serum,1% penicillin/streptomycin and 1% glutamine),mixed severaltimes and seededat a density of 150-300,000cells/80 cm2 tissueculture flask. Cells were fed every 24-48 hours and reached confluence after 7-12 days. Cells were used between the secondand tenth passages. Cells were harvestedandmembranespreparedasdescribedby Anand-Srivastava et al. (1). At confluence, the cells were rinsed 2 times with PBS and scrapedwith a rubber policeman in 10 mls of PBS. The cells were centrifuged at 1500 x g for 5 minutes and resuspendedin homogenization buffer containing 10 mM Tris, 1 mM EDTA, and 1 mM DTT, pH 7.5. The cells were homogenized in a Type A Dounce homogenizer for 10 strokes. The homogenatewas centrifuged at 5000 x g for 10 minutes. The supematant was discardedand the pellet was hand homogenizedwith a Teflon-Glass homogenizeron ice in 1 ml of homogenizationbuffer. The final protein concentrationwas approximately 2 mg/ml. Protein was determinedaccordingto Lowry (4). Adenvlate cvclase assay: Adenylate cyclase activity wasmeasuredasdescribedpreviously (5). The incubation buffer contained 200 uM alpha-[32P]ATP (50 cpmfpmol), 30 mM Tris-HCl (pH 7.5), 1 mM MgC12,O. 1 mM cyclic AMP, 0.1% bovine serumalbumin, 10 mM creatinephosphate,10U/ml creatinephosphokinase,1 mM DIT, 1.Oug/ml adenosine deaminaseand the indicatedadditionsin a final volume of 100ul. The time and protein dependencefor enzyme activity are shown in Figure 1. Formation of cyclic AMP was linear over a thirty minute time period and with increasing protein concentration (0.20-0.75 t&11). Accordingly, in subsequentstudies,adenylate cyclase assayswere conducted for 15 minutes with 0.20-0.50 ug/ul membrane protein concentration. Assays were initiated by the addition of membraneand terminated by the addition of 1% SDS. The experiments were performed in a shaking water bath at 37°C. [32Pl Cyclic AMP was purified by sequentialchromatography on Dowex and alumina columns as described by Salomon (6). [3H] Cyclic AMP was added as a recovery standard. Statistical analysis: Data were analyzed by paired t-test. All resultsare expressedasmean + SEM. Statistical significance wasset at PcO.05.

ij !, A b b

30 30

10

cpd % k 1 23

8

20 15

4

10

5 0

0

2

10

20

30

0

TLLlE (mid

2s

So PRomN

75

lrn" lot

(up)

Figure 1. Effect of time and protein concentration on basal and sodium fluoride stimulated adenylate cyclase activity iu membranes obtained from cultured porcine coronary vascular smooth muscle cells. The data were obtained from one experiment performed in duplicate.

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Chemicals: CGS 15943A was a generousgift from Ciba-Geigy (Summit, New Jersey). Adenosine analogswere obtainedeither from Boehringer-Mannheim (Indianapolis,IN) or ResearchBiochemicals Incorporated (Natick, MA). Radiochemicalswere obtained from New England Nuclear (Boston, MA). All other chemicals were obtained from Sigma ChemicalCompany (St. Louis, MO).

RESULTS AND DISCUSSION In order to determine whether Al adenosinereceptor mediatedinhibition of adenylate cyclase was present in porcine coronary vascular smoothmuscle cells, we examined the effect of lo-1OM to lo-5M PIA, an Al adenosine receptor agonist, on Gpp(NH)p stimulatedenzyme activity (data not shown). As expected, Gpp(NH)p (0.1-100 uM) alone causeda concentration dependentincreasein adenylate cyclase activity. However, PIA (lo-1OM to lo-6M) did not affect adenylate cyclase activity at either a submaximal (0.1 mM) or maximal (100 n&I) stimulatory concentration of Gpp(NH)p (Table 1). A further increasein the PIA concentrationto 10-SM led to a significant potentiation of 0.1 mM and 100 mM Gpp(NH)P stimulatedadenylate cyclase activity (PcO.05) (Table 1). A two-fold augmentationof 0.1 mM Gpp(NH)P stimulatedadenylatecyclase activity was observedat 1 mM PIA (Table 1). At 100 mM Gpp(NH)P, PIA (10-5-10-3 M) significantly stimulated

TABLE

1

EFFECTOFPL4ON GPP(NH)PSTIMULATED ADENYLATE CYCLASEACTlVrrY (PMOLES/MIN/MG) IN CULTUREDPORCINECORONARYVASCULAR SMOOTH MUSCLECELLS

CONDlTION

GPP(NH)PCONC. (uM) 0.1

100

CONTROL

4.3kO.6

31.1k7.6

+PlA (IO-‘M)

4.4kO.7

36.229.3

+PIA (lo-*M)

4.2kO.6

35.Q8.8

+PIA ( 10-7M)

4.1kO.7

36.Q8.9

+PIA (10-6M)

4.4kO.7

35.8k9.0

+PIA ( 10-5M)

5.3&0.8*

37.9;t9.3*

DMSOCONTROL

4.8kO.8

45klO.l

+PL4 ( 10-4M)

7.8+1.6#

55.0+11.4#

+PIA (1O”M)

9.2+2.3#

56.1+10.1#

Thevaluesrepresent themean+ SEM for 8 experimptsat 0.1 uM GPP(NH)Pand6 at 100uM GPP(NH)P. * = P < 0.05versuscontrol; = P < 0.05versusdmsocontrol. 1299

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adenylate cyclase activity aswell. Thus, at higher concentrations,PIA behaved asan A2 adenosinereceptor agonist. Since PIA alone failed to inhibit Gpp(NH)p stimulated adenylate cyclase activity, we also studied the effect of both PIA and CPA (another Al agonist), on Gpp(NH)p stimulatedadenylate cyclase activity in the presenceof a selective A2 adenosinereceptor antagonist(i.e.; CGS 15943A (7)). A concentration of 1 nM PIA and CPA were utilized for this set of experiments 1) to avoid stimulation of adenylate cyclase which occurs at higher concentrations of PIA (Table 1) or CPA, 2) becauseadenosineanalog mediated inhibition of adenylatecyclaseactivity via the Al adenosinereceptor typically exhibits IC50 values in the nanomolar range (8), and, 3) since nanomolar cyclohexyladenosine, an Al selective agonist,hasbeendemonstratedto causevasoconstrictionof arteriolesin hamster skin (8). CGS 15943A concentrations of 3 and 30 nM were used in these experiments since CGS 15943A displayed an IC50 of 3 nM in blocking [3H]5’-Nethylcarboxamidoadenosinebinding to rat striatal A2 adenosinereceptors and 18 nM in blocking adenosinestimulatedadenylatecyclase activity in guineapig synaptoneurosomes (7). We recognize that CGS 15943A exhibits an IC50 of 20 nM in preventing [3H]cyclohexyladenosine binding to rat forebrain Al adenosine receptors (7) and is therefore a selectiveand not totally specificantagonistof the A2 adenosinereceptor. In the presenceof either 3 or 30 nM CGS 15943A, PIA (1 nM) failed to inhibit Gpp(NH)p stimulation of adenylate cyclase. CPA (lo-9M) alone, also failed to inhibit 0.1 uM Gpp(NH)p stimulatedadenylate cyclase activity. However, in the presenceof 30 nM CGS 15943A, CPA produceda significant inhibition of adenylatecyclaseactivity. The ability of CPA to inhibit adenylatecyclase activity was dependenton the absolutelevel of Gpp(NH)p stimulated adenylate cyclase activity.

Thus in four experiments (hereafter

referred to asGroup I) in which Gpp(NH)p stimulatedadenylate cyclase from 1.2&O.1 to 2.3kO.4 pmoles/min/mg, 1 nM CPA alone or with 3 or 30 nM CGS 15943A failed to inhibit adenylate cyclase activity (Figure 2A). In contrast, in four other experiments (hereafter referred to as Group II) in which Gpp(NH)p stimulatedadenylate cyclase from

A.

GROUP I

8.

GROUP II

C.

GROUP II

5

35 0 GPP(NH)P IGPP+cPA(l

(0.1

uU) nM)

30 f :;I 15 5

f

10 I

2i

5 0

-at

0 0

3

30

0

CGS

3

15943A

Jo

CONC.

0

3

30

(nM)

Figure2. Al adenosine receptormediated inhibitionof adenylate cyclaseactivity in culturedporcine coronaryvascularsmoothmusclecellsis shownherefor GroupI (panelA) andGroupII (panelB). In panelC, percentinhibitionof Gpp(NH)pstimulated adenylatecyclaseby 1 nM CPA(mean+ SEM)is shownfor GroupII. 1300

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3.8k1.4 to 6.1k1.6, 1 nM CPA causeda gradedincreasein inhibition of adenylate cyclase with increasingconcentration of CGS 15943A (Figure 2B). Thus, the absolute,rather than the fold augmentation, of Gpp(NH)p stimulated adenylate cyclase activity was most important in demonstrating inhibition by CPA. The percent inhibition of Gpp(NH)p stimulatedadenylatecyclase activity by 1 nM CPA in Group II is shown in Figure 2C. At 3 and 30 nM CGS 15943A, 1 nM CPA causeda 21+3% and 27+3% (PcO.05) inhibition of adenylatecyclaseactivity, respectively. The Al adenosinereceptor is characterizedby its ability, when stimulated,to inhibit adenylate cyclase activity. In contrast, activation of the A2 adenosinereceptor causes stimulation of adenylate cyclase activity (9). The potency seriesfor activation of the Al adenosinereceptor is PIA > adenosine> NECA and the converseis characteristicof the A2 adenosinereceptor (9). In an intact canine model, Kusachi et al. (2) demonstrated a potency order of adenosineanalogsassociatedwith the A2 adenosinereceptor. However, they also found a potency order of R-PIA > adenosineand stereoselectivity of PIA characteristic of the Al adenosinereceptor (2). Thus, it was suggestedthat the adenosine receptor on vascular smoothmuscleis a hybrid (2) becauseit sharedpropertiesof both Al and A2 adenosine receptors. Data obtained in the present study are consistent with observations made by Kusachi et al. (2) in that evidence of both Al and A2 adenosine receptor effects were present in cultured vascular smooth muscle cells obtained from porcine coronary arteries. There is evidence to suggestin certain vascular beds that adenosinecan cause vasoconstriction via a high affinity Al adenosinereceptor. Thus, adenosineis known to causerenal vasoconstriction andinhibition of renin secretionpresumablyvia Al adenosine receptors (10). In a recent study, Al adenosinereceptor mediated vasoconstriction also was demonstratedin subcutaneousarterioles of the hamster(8). We have extended these findings by demonstratingthe inhibition and stimulation of adenylate cyclase in an single cell type, namely cultured coronary vascular smooth muscle cells. Accordingly, it is reasonable to suggest that activation of the Al adenosine receptor in the coronary circulation could competewith vasodilation related to A2 adenosinereceptor stimulation and thereby influence the regulation of myocardial blood flow. The Al adenosinemediated inhibition of adenylate cyclase activity in coronary vascular smooth muscle cells was relatively modest in the present study. It remains possible under certain conditions that the Al adenosinereceptor signalmay be enhanced. Thyroid hormone status,caffeine ingestion, pregnancy/lactation and aging have all been demonstratedto play a role in the regulation of the adenosinereceptor (11). For example, caffeine treated rats display increased sensitivity of the Al adenosinereceptor to PIA mediatedinhibition of adenylatecyclaseactivity in rat brain ascomparedto control animals (12). Thus, it is possible that the weak Al adenosinereceptor signaling in coronary vascular smoothmuscle cells observed in the present study may be enhancedunder other conditions. Additional experimentsare requiredto test this hypothesis. Although the Al adenosinereceptor agonistsPIA and CPA have similar IQ0 values for inhibition of adenylatecyclaseactivity in fat cells(9), we observeddifferencesin 1301

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their ability to inhibit adenylate cyclase activity in cultured vascular smoothmusclecells. Unlike CPA, inhibition of adenylate cyclase activity by PIA was not observed in the presenceof CGS 15943A, a selective A2 adenosinereceptor antagonist. The difference in behavior of PIA and CPA is not completely understood. However, it may reflect structural differences between PIA and CPA and their interaction with CGS 15943A at Al and A2 adenosinereceptor sites. It shouldbe noted that although the IC50 value for inhibition of fat cell adenylate cyclase activity is nearly identical for the two analogs,the Ki value of 0.32 nM for CPA inhibition of [3H]cyclohexyladenosine binding to Al adenosine receptorsin rat brain membranesis nearly 4 fold lower than for PIA (9). Furthermore, in pheochromocytoma PC12 cells, the EC50 of 980 nM for PIA activation of adenylate cyclase activity via A2 adenosinereceptorsis 3 fold lower than that observed with CPA (9). Thus, as observed in the present studies, there is evidence from previous studies demonstratingdifferencesin the actionsof the two Al adenosinereceptor agonistsPIA and CPA. In summary,cultured vascular smoothmusclecells obtainedfrom porcine coronary artery exhibit Al adenosinereceptormediatedinhibition of adenylatecyclaseactivity. This suggeststhat in addition to its well known vasodilator effects, adenosinealsomay promote vasoconstrictionin coronary vasculatureunder certain conditions. Acknowledzments: Supportedin part by grantsfrom the USPHS NIH-NHLBI [HL 299511and from the American Heart Association,Rhode IslandAffiliate. Dr. Gewirtz is an EstablishedInvestigator of the American Heart Association. The authorsacknowledge the secretarialassistance of ChristineAbatiello andthe expert technicalassistance of Ray Boynton. REFERENCES 1. 2. 3. 4. 5. 6. 7. i: 10. :;:

Anand-Srivastava, M.B., Franks, D.J., Cantin, M., and Genest, J. (1982) Biochem. Biophys. Res. Comm. 108: 213-219. Kusachi, S., Thompson, R.D., and Olsson, R.A. (1983) J. Pharmacol. Exp. Ther. 227: 316-321. Gunther, S., Alexander, R.W., Atkinson, W.J., and Gimbrone, M.A. (1982) J. Cell Biol. 92: 289-298. Lowry, O.H., Rosebrough, N.J., Farr, A.L., and Randall, R.J. (195 1) J. Biol. Chem. 193: 265-275. Mills, I., Garcia-Sainz, J.A., and Fain, J.N. (1986) B&hem. Biophys. Acta 876: 619-630. Salomon, Y. (1979) Adv. Cyclic Nucleotide Res. 10: 35-55. Williams, M., Francis, J., Ghai, G., Braunwalder, A., Psychoyos, S., Stone, G.A., and Cash,W.D. (1987) J. Pharmacol.Exp. Ther. 241: 415-420. Stojanov, I. and Proctor K.G. (1989) Circ. Res. 65: 176-184. Ukena, D., Olsson, R., and Daly, J.W. (1986) Can. J. Physiol. Pharmacol. 65: 365-376. Murray, R.D. and Churchill, P.C. (1985) J. Pharm. Exp. Therap. 232: 189-193. Stiles, G.L. (1986) Trends in Pharmacol.Sci. 6:486-490. Green, R.M. and Stiles, G.L. (1986) J. Clin. Inv. 77:222-227.

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